The present invention relates to purifying the protein human serum albumin (HSA) extracted from serum or recombinant human albumin (rHA) produced by transforming a microorganism with a nucleotide coding sequence encoding the amino acid sequence of human serum albumin. In this specification, the term xe2x80x9calbuminxe2x80x9d refers generically to HSA and/or rHA.
Albumin is used to treat patients with severe bums, shock or blood loss. It is also used to supplement media used for growing higher eukaryotic cells and as an excipient in the formulation of therapeutic proteins. At present, the demand for the product is satisfied by albumin extracted from human blood. Examples of extraction and separation techniques include those disclosed in: JP 03/258 728 on the use of a cation exchanger; EP 428 758 on the use of anion exchange followed by cation exchange; and EP 452 753 on the use of heating, adding salt and diafiltering.
The production of rHA in microorganisms has been disclosed in EP 330 451 and EP 361 991. Purification techniques for rHA have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants: and EP 319 067, alkaline precipitation and subsequent application of the rHA to a lipophilic phase having specific affinity for albumin.
The present invention relates to a process for purifying the protein human serum albumin extracted from serum or recombinant human albumin produced by transforming a microorganism with a nucleotide coding sequence encoding the amino acid sequence of human serum albumin. The present invention provides highly purified albumin.
The present invention provides highly purified albumin.
One aspect of the present invention provides a process for purifying albumin, the process comprising the steps of applying a relatively impure albumin solution to a chromatographic material for which the albumin has no specific affinity such that albumin binds to the material, and eluting the bound albumin from the material by applying a solution of a compound having a specific affinity for albumin. Preferably, the chromatographic material is a cation exchanger, such as SP-Sepharose FF, SP-Spherosil etc, as listed below in Example 2. The compound with specific affinity for albumin may be octanoate (eg sodium octanoate), other long chain (C6 to C22) fatty acids, salicylate, octylsuccinate, N-acetyltryptophan or a mixture of two or more of these.
A second aspect of the invention provides a process for purifying albumin, the process comprising the steps of subjecting an albumin solution to cation exchange chromatography in which the albumin is bound to a cation exchange material and then anion exchange chromatography in which the albumin is bound to an anion exchange material.
The albumin which is eluted from the cation exchange material may be subsequently treated by one or more of affinity chromatography, ultrafiltration and gel permeation before being subjected to the said anion exchange chromatography. Hence, in a preferred embodiment, the process comprises the steps of:
(a) passing an albumin solution through a cation exchange matrix under conditions such that the albumin will bind to the matrix:
(b) eluting from said matrix an albumin-containing cation exchange eluate:
(c) passing said eluate through an affinity matrix comprising an albumin-binding compound:
(d) eluting from said matrix an albumin-containing affinity matrix eluate:
(e) passing said eluate, optionally after ultrafiltration, through a gel permeation matrix to obtain a fraction enriched in albumin;
(f) passing the said albumin-enriched fraction through an anion exchange matrix under conditions such that albumin will bind to the matrix: and
(g) eluting from said anion exchange matrix a purified albumin-containing product.
Alternatively, the albumin which is eluted from the cation exchange material may be applied to the said anion exchange material without any intervening treatment (other than dilution). Hence, a second preferred embodiment provides a process for purifying albumin, comprising the steps of:
(a) passing an albumin solution through a cation exchange matrix under conditions such that the albumin will bind to the matrix;
(b) eluting from the matrix an albumin-containing cation exchange eluate;
(c) passing the cation exchange eluate through an anion exchange matrix under conditions such that the albumin will bind to the matrix;
(d) eluting from the anion exchange matrix an albumin-containing anion exchange eluate;
(e) passing the anion exchange eluate through an affinity matrix comprising an albumin-binding compound;
(f) eluting from the affinity matrix an albumin-containing affinity matrix eluate;
(g) passing the affinity matrix eluate through a gel permeation matrix to obtain a fraction enriched in albumin.
Preferably, prior to the cation exchange step, the albumin solution is conditioned by adding octanoate and/or other albumin stabiliser (eg sodium acetyltryptophanate) thereto to a final concentration of from about 1-10 mM and adjusting the pH to about 4.0-5.0.
Advantageously, the albumin retained in the cation exchange step is washed with a high salt solution (eg 0.5-2.0 M NaCI buffered at pH 4.0 with 10-100 mM, preferably 20-40 mM for example 27 mM sodium acetate) before being eluted.
Preferably, in processes in which the cation exchange eluate is passed directly to the anion exchanger, the albumin is eluted in the cation exchange step using a buffer containing a compound having a specific affinity for albumin, especially an acid or salt thereof, for example octanoate or any other long chain (C6-C22) fatty acid. salicylate, octylsuccinate or N-acetyltryptophan.
Suitably, the albumin is eluted from the anion exchanger with a buffer containing a high level (eg at least 50 mM, preferably 50-200 mM, for example 80-150 mM) of a boric acid salt, for example sodium or potassium tetraborate.
The albumin purified in accordance with the invention may then, with or without intervening process steps, be subjected to chromatography on a resin containing an immobilised compound which will selectively bind glycoconjugates and saccharides, such as aminophenylboronic acid (PBA).
In any process of the invention which involves affinity chromatography, the affinity chromatography preferably uses a resin comprising an immobilised albumin-specific dye, such as a Cibacron Blue type of dye, preferably immobilised on the resin via a spacer such as 1,4-diaminobutane or another spacer of C1-8, preferably C1-6, eg C1-5 and most preferably C4 length, preferably having xcex1,xcfx89-diamino substitution. Surprisingly, we have found that such dyes actually have a greater affinity for a 45 kD albumin fragment which can be produced in cultures of HA-secreting microorganisms, than they do for the full length albumin molecule. The 45 kD fragment typically consists of the 1-403 to 1-409 region and is disclosed in Sleep et al (1990) Bio/Technology 8, 42-46 and in WO 95/23857.
The purified albumin solution prepared by the process of the invention may be further processed according to its intended utility. For example, it may be ultrafiltered through an ultrafiltration membrane to obtain an ultrafiltration retentate having an albumin concentration of at least about 80 g albumin per litre, with the ultrafiltration retentate being diafiltered against at least 5 retentate equivalents of water. It can be advantageous to include ammonium ions in certain chromatographic steps, for example in the step involving immobilised aminophenylboronate. Surprisingly, we have found that such ammonium ions are relatively tightly bound to the albumin. It is preferable for such ammonium ions to be removed from the albumin and we have found that this can be achieved by use of a counter-ion. The desirability of exposing the albumin to a counter-ion would not have occurred to those in this art since prior processes have not involved ammonium ions and there was no reason to suppose that ammonium ions would be bound by the albumin. Accordingly, a further aspect of the invention provides a method of purifying an albumin solution comprising exposing the solution to a solution of a counter-ion such that ammonium ions are displaced from the albumin and can be removed from the solution.
The counter-ion (preferably a metal ion such as sodium ions) can be added to the albumin solution and the ammonium ions removed by dialysis, or the ammonium ion can be diafiltered away across a semi-permeable membrane separating the albumin from the solution of the counter-ion, or they can be removed by gel permeation chromatography. Diafiltration against at least five retentate volumes of 50 mM sodium chloride is generally suitable.
The albumin obtained has been found to have extremely low levels of, or to be essentially free of, colorants, lactate,; citrate, metals, human proteins such as immunoglobulins, pre-kallikrein activator, transferrin, xcex11-acid glycoprotein haemoglobin and blood clotting factors, prokaryotic proteins, fragments of albumin albumin aggregates or polymers, endotoxin, bilirubin, haem, yeast proteins and viruses. By xe2x80x9cessentially freexe2x80x9d is meant below detectable levels. The term xe2x80x9ccolorantxe2x80x9d as used herein means any compound which colours albumin. For example, a pigment is a colorant which arises from the organism, especially yeast, which is used to prepare recombinant albumin, whereas a dye is a colorant which arises from chromatographic steps to purify the albumin. At least 99%, preferably at least 99.9%, by weight of the protein in the albumin preparations purified by the process of the invention is albumin. Such highly pure albumin is less likely to cause adverse side effects.
The albumin produced by the process of the invention has been found to be at least 99.5% monomeric, preferably substantially 100% monomeric by reducing SDS PAGE, and is characterised by one or more of the following characteristics. It has an aluminium ion content of less than 150 ng, preferably less than 100 ng; an iron ion content of less than 3,000 ng, preferably less than 1,000 ng; a copper ion level of less than 10,000 ng, preferably less than 5,000 ng; a magnesium ion level of less than 3,000 ng, preferably less than 1,500 ng; a zinc ion level of less than 5,000 ng, preferably less than 3,000 ng, a manganese ion level of less than 50 ng, all based on one gram of albumin; a glycation level of less than 0.6, preferably less than 0.15 (more preferably less than 0.05), moles hexose/mole protein; a level of low molecular weight contaminants of below 20 V.sec, preferably less than 10 V.sec, measured as in Example 9 below; a single peak on a capillary zone electrophoretogram: intact, ie homogeneous, C-terminus and N-terminus: a free thiol content of at least 0.85 mole SH/mole protein; and no more than 0.3 mol/mol of C10 to C20 fatty acids and substantially no C18 or C20 fatty acids.
The starting material may be an albumin-containing fermentation medium, or the impure albumin solution may be a solution obtained from serum by any of the plethora of extraction and purification techniques developed over the last 50 years. for example those disclosed in Stoltz et al (1991) Pharmaceut. Tech. Int. Jun. 1991. 60-65 and More and Harvey (1991) in xe2x80x9cBlood Separation and Plasma Fractionationxe2x80x9d Ed. Harris, Wiley-Liss, 261-306.
Especially when the albumin is rHA produced in protease-deficient yeasts or other organisms, the process does not normally comprise a heat treatment step as part of the purification process (in contrast to EP 428 758 and EP 658 569). Similarly, if it is prepared from microorganisms (rather than from humans) it does riot normally require a final pasteurisation step (typically 60xc2x0 C. for one hour).
The final product may be formulated to give it added stability. Preferably, the highly pure albumin product of the invention contains at least 100 g, more preferably 1 kg or 10 kg of albumin, which may be split between a plurality of vials.
Although the process of the present invention can be utilised to obtain more purified albumin from an impure albumin solution from a number of sources, such as serum, it is particularly applicable to purifying recombinant human albumin (rHA). The albumin produced in accordance with the invention may be any mammalian albumin, such as rat, bovine or ovine albumin, but is preferably human albumin. DNA encoding albumin may be expressed in a suitable host to produce albumin. Thus, DNA may be used in accordance with known techniques to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of albumin. Such techniques include those disclosed in EP-A-73 646. EP-A-88 632. EP-A-201 239 and EP-A-387 319.
Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae. Pichia pastoris and Kluyveromyces lactis), filamentous fungi (for example Aspergillus), plant cells. animal cells and insect cells. The preferred microorganism is the yeast Saccharomyces cerevisiae. 
Exemplary genera of yeast contemplated to be useful in the practice of the present invention are Pichia (Hansenula), Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Preferred genera are those selected from the group consisting of Pichia (Hanseniula), Saccharomyces, Kluyveromyces, Yarrowia and Hansenula. Examples of Saccharomyces spp. are S. cerevisiae. S. italicus and S. rouxii. Examples of Kluyveromyces spp. are K. fragilis and K. lactis. Examples of Pichia (Hansenula) are P. angusta (formerly H. polymorpha), P. anomala. P. pastoris and P. capsulata. Y. lipolytica is an example of a suitable Yarrowia species.
It is advantageous to use a yeast strain which is deficient in one or more proteases. Such strains include the well-known pep4-3 mutants and strains with mutations in the PRA1 and/or PRB1 genes, as in Woolford et al (1993) J. Biol. Chem. 268, 8990-8998, Cabezon et al (1984) P.N.A.S. 81, 6594-6598, EP-A-327 797 and Jones et al (1982) Genetics 102, 665-677. Alternatively, the proteases in the fermentation medium may be inactivated by heating. The existence of proteases reduces the yield of the albumin during the overall process. Preferably, the yeast has a low (or zero) level of the Yap3p protease and/or of the hsp150 heat shock protein, for example as a result of having the respective genes disrupted, as is taught in our patent applications published as WO 95/23857 and WO 95/33833. respectively. Yap3p can cause the formation of the 45 kD albumin fragment referred to below, and hsp150 co-purifies with albumin in some separation steps.
Yeast may be transformed with an expression plasmid based on the Saccharomyces cerevisiae 2xcexcm plasmid. At the time of transforming the yeast, the plasmid contains bacterial replication and selection sequences, which may be excised, following transformation, by an internal recombination event in accordance with the teaching of EP 286 424. The plasmid may also contain an expression cassette comprising: a yeast promoter (such as the Saccharomyces cerevisiae PRB1 promoter), as taught in EP 431 880: a sequence encoding a secretion leader, for example one which comprises most of the natural HSA secretion leader, plus a small portion of the S. cerevisiae xcex1-mating factor secretion leader as taught in WO 90101063: the HSA coding sequence obtainable by known methods for isolating cDNA corresponding to human genes, and also disclosed in, for example, EP 73 646 and EP 286 424; and a transcription terminator, for example the terminator from Saccharomyces ADH1, as taught in EP 60 057.
The choice of various elements of the plasmid described above is not thought to be directly relevant to the purity of the albumin product obtained although the elements may contribute to an improved yield of product.
Preferred aspects of the invention will now be described by way of example and with reference to the accompanying drawings, in which: